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Baculovirus Production with Bac-to-Bac (Invitrogen)
By: Scott Hansen BACMID → Baculovirus → SF9 protein expression Making DH10 Bac competent cells 1. Cell line was obtained from Matt Good in the Lim Lab. 2. Grow single colony of DH10 Bac in 5mL of LB plus 50ug/mL KAN and 10ug/mL tetracycline overnight at 37C (shake 250ppm). 3. Inoculate 400mL of LB/TET/KAN. Grow at 37C to OD590 of 0.375. 4. Harvest cells (8X 50mL conicals). Centrifuge 4000xg, 4C for 15min. 5. Resuspend in 10mL of ice cold CaCl2 solution (60mM CaCl2, 15%(v/v) glycerol, 10mM PIPES, pH 7 with KOH. Filter sterilize). 6. Centrifuge 15 min cold. 7. Resuspend in 10mL of CaCl2 solution. Incubate on ice for 30 minutes. 8. Centrifuge 15 min cold. 9. Resuspend each pellet in 2mL of CaCl2 solution. 10. Aliquot and freeze with liquid N2. Note: We now use the Zymo kit for making competent cells. Follow those directions for DH10Bac cells. Plates for making BACMID 10g tryptone, 5g yeast extract, 5g NaCl, 15g agar per liter of water. Autoclave for 30 min. Add to LB plates post autoclave: Final concentrations 50ug/mL Kanamycin (stock= 50 mg/mL in water) 10ug/mL Tetracycline (stock=10mg/mL in ethanol, store in -20C, should be a bright yellow sol’n) 7ug/mL Gentamycin (stock=7 mg/mL in water) **Oct 14 - stock is 50 mg/mL 40ug/mL X-GAL (stock=40mg/mL in DMF) 40ug/mL IPTG (stock=238mg/mL or 1M stock in water) store plates in dark cold room for up to 2 months. DH10 BAC transformation 1. Transform 1ng (~5µL) plasmid into 50µL of DH10 alpha comp cells (Rack 2-1, bottom shelf, third in) 2. Ice 30 minutes 3. Heat shock 4. Recover in LB, TPM, or SOC for 4 hours at 37C. 5. Plate on BACMID plates ** Only plate 1-20 uL of culture onto plates. 6. Grow at 37C for 2 days. 7. Isolate white colonies and restreak onto BACMID plate. (optional) 8. Freeze BACMID strain -80C in media containing 10% glycerol. Isolation of BACMID 1. Inoculate 5mL LB containing 50ug/mL Kanamycin, 10ug/mL Tetracycline, 7ug/mL Gentamycin. Grow at 37C overnight. 2. Spin 4mL culture. Remove supernatant. 3. Add 300µL of solution I (15mM Tris HCl pH8, 10mM EDTA, 100µg/mL RNase) *Note this is the same as Qiagen or most miniprep kits buffer #1 4. Add 300µL of solution II (0.2N NaOH, 1% SDS).gently mix. *Same as buffer #2 from miniprep kits 5. Incubate for 5 minutes. 6. Slowly add 300µL of 3M K+Acetate pH5.5. White ppt containing protein and gDNA will form. Mix well by inverting the tube several times. Incubate on ice for 5-10 minutes. *Same as buffer #3 from miniprep kits 7. Centrifuge for 10 minutes, 14000xg or max speed. Use fuge in first left cold room if possible, it has larger rotor, more force to pellet insoluble protein. 8. Avoid white ppt. And remove the supernatant to a fresh 2mL tube. Combine supernatant with 800µL of isopropanol and mix well. Ice for 5-10 minutes. At this stage the sample can be stored overnight in -20C if desired. 9. Centrifuge for 15 min, 14,000 x g or max speed. Off white pellet should be seen. This is the bacmid DNA and leftover protein. Less white (protein) is better. 10. Remove supernatant and discard it. Add 0.5mL of 70% ethanol gently to the DNA pellet. Careful not to disturb pellet. 11. Centrifuge for 15 min, 14,000 x g. 12. Remove supernatant. Air dry. CRITICAL, MUST BE SURE ALL THE ETHANOL IS EVAPORATED. PELLET SHOULD TURN MOSTLY CLEAR TO INVISIBLE WHEN FULLY DRY. MAY TAKE 30-60 MIN. 13. Dissolve DNA in 40µL of sterile water. Do not pipette vigorously, you can break the bacmid DNA because it is very large. Measure concentration and purity on the nanodrop. Store in -20C. PCR confirmation 1. Use 1µL of BACMID in 50µL PCR with Expand polymerase. 2. Use internal primers and M13/pUC forward and reverse. 1µL of 15pmol/µL primers. a. M13/pUC FW(-40bp) GTTTTCCCAGTCACGAC b. M13/pUC FW(-20bp) GTAAAACGACGGCCAG c. M13/pUC RV CAGGAAACAGCTATGAC Growing SF9 cells Thaw a vial of 10^106 cells quickly at 37. Spray clean outside of vile with 70% ethanol. Dilute cells into 10mL of fresh media and start shaking. Monitor cells every day and split the cells when they reach 5-8^106/mL. Keep between 0.5-8.0 x 106 cells/mL SF900 II SFM (Cat# 10902) (serum free media) Penicillin; Streptomycin (use at 0.5-1x) Grow at 27C, humidified, no extra CO2 Cells need to be well aerated. Grow 200mL in 500mL spinner flask; 400mL in 1 liter. Transfecting SF9 cells : ''' Treat each well in a 6-well dish as a sterile environment! Change tips for each well every time. ''' 1. The night before, split cells to 1x106 cells/mL. 2. Next day, count cells. Should be between 1.8-2.2x106 cells/mL. 3. Plate 0.5-1 x 106 cells in 6-well plate (found that plating 0.5mil/ml turns out to be a better density). NOTE: Must '''mix cells by doing figure-8 movement of whole plate. Cells MUST be evenly dispersed in the well!!' * '''Optional: '''plate 2 different densities of cells just in case. Plate calculated 1 million and 0.5 X of that to give yourself a backup in case density if off. 4. Cells seem to adhere really quickly. I would check them after 10-15 min (about 30min seems sufficient, then wash into no A/A media(step 7)). Check cell density in microscope to make sure it’s not too dense or too low. Cells should be evenly spaced but not touching each other very much. They need room to divide as they take up the DNA during division. If most cells are already touching each other, they are too dense and replate at a lower density. 5. Combine 1-5µg (~5µL @ 200ng/uL) BACMID DNA + 6µL CellFectin (invert to mix) + 200µL media (NO PEN/STREP). 6. Incubate CellFectin/BACMID DNA mixture for 60 minutes at 25C. 7. Wash cells 2x with SF900 II media (NO PEN/STREP) 8. Combine 800µL media (NO PEN/STREP) and 200µL CellFectin/BACMID DNA mixture (1mL final). 9. Add 1mL transfection mixture to cells. 10. Replace with at least 2mLs A/A SF900 II media after ~5 hours of transfection in 27C. '''P1 titer' 1. After 4-6 days the cells will begin sluffing off the plate. Have a control monolayer for comparison. 2. Remove P1 media into sterile 2mL tube(should be approx. 2 mLs total). 3. Spin full speed for 3 minutes in table top centrifuge. 4. Transfer supernatant to a new sterile 2mL tube. Store in dark cold room. 5. Virus in media containing 2% FBS will protect against proteases and can be frozen in -80C. (Add the FBS to media first and sterilize!) P2 titer 1. One or two days before, split cells to 1x106 cells/mL (100mLs is sufficient) for P2. 2. Next day, count cells to be between 1.8x106-2.2x106 cells/mL. 3. Inoculate 20 or 50 uLs of P1 stock directly into the P2 cells. 4. Incubate for 5 to 7 days(be sure to count cells each day to be sure that growth is arrested by the spreading viral infection). Cells should arrest after 2 to 3 days. or 5. Spin down total volume of cells using GSA rotor at max speed. 6. Add FBS to 0.5%. Then sterile filter the supernatant using vacuum. (Important to add FBS before filtering! Mark on FBS cap so others know the FBS was opened outside the hood). 7. Collect media, cap in hood, store supernatant in dark cold room. 8. Use P2 for protein production at 1:100 dilution.